ΒETA-GLUCAN EXTRACTION FROM WASTE … SACCHAROMYCES CEREVISIAE USING AUTOLYSIS, ENZYME, ULTRASONIC...

American Journal of Research Communication www.usa-journals.com

OPTIMIZATION OF ΒETA-GLUCAN EXTRACTION FROM WASTE BREWER’S YEAST SACCHAROMYCES CEREVISIAE USING AUTOLYSIS, ENZYME,

ULTRASONIC AND COMBINED ENZYME – ULTRASONIC TREATMENT

Tran Minh Tam1, Nguyen Quoc Duy2, Nguyen Phuoc Minh1, Dong Thi Anh Dao1

1 Dept. food Technology, Vietnam National Uni. HCMC University of Technology, Vietnam 2 Dept. Chemical and Food Tech, Nguyen Tat Thanh University, Vietnam

* Contact email: [email protected] [email protected]

ABSTRACT

Beta-glucans are complex, high molecular (100 – 200 kDa) polysaccharides, found in the cell wall of many yeasts and cereals. Yeast beta-glucans differ from their cereal counterparts in that they comprise a mixture of beta-1,3- and 1,6-glucans, compared to the cereal derivatives which are a mixture of beta-1,3- and 1,4-glucans. In this study, beta-glucan was prepared from waste beer yeast by enzymatic, ultrasonic and combined method. In the ultrasound-assisted extraction, estimated optimum conditions were as follows: treatment time of 11.91 minutes. Cell disruption yield of beta-glucans has inferior recovery enzyme method. Maximum cell disruption yield by enzyme-ultrasonic is 72.06% at power 28,9w/g.

Keywords: β-glucan, Saccharomyces cerevisiae, enzymatic hydrolysis, sonication

{Citation: Tran Minh Tam, Nguyen Quoc Duy, Nguyen Phuoc Minh, Dong Thi Anh Dao. Optimization of beta-glucan extraction from waste brewer’s yeast Saccharomyces cerevisiae using autolysis, enzyme, ultrasonic and combined enzyme – ultrasonic treatment. American Journal of Research Communication, 2013, 1(11): 149-158} www.usa-journals.com, ISSN: 2325-4076.

1. INTRODUCTION

The natural sources of β-glucans are bacteria, yeast, algae, mushrooms, barley as well as oat. Th e native chemical structure of β-glucans depends on the source they are isolated from. Each type of β-glucan, generally derived from different sources, has an unique structure in which glucose units are linked together in different ways (Stone and Clarke, 1992; Stone, 2009). β-Glucans from different sources have different chemical structures [10].

Tam, et al., 2013: Vol 1(11) [email protected] 149

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Figure 1. Structure of the cell wall of yeast Saccharomyces cerevisiae (Osumi, 1998).

Figure 2. Chemical structure of β-glucan in yeast Saccharomyces cerevisiae (Kath, 1999).

Yeast is a well known microorganism that is used in biotechnology since ancient times [2]. Therefore it is a good source of β-glucan. β-Glucans in yeast cell walls are branch-onbranch molecules containing linear (1,3)-β-glucosyl chains that are joined through (1,6)-linkages (Osumi, 1998; Kath and Kulicke, 1999; Clavaud et al., 2009) (Figure 1). These molecules occur as complexes with other polysaccharides and proteins (Osumi, 1998) [5].

β-Glucans, polymers of glucose linked by β-glycosidic bonds, are widely distributed in the cell walls of microorganisms, mushrooms and plants. β-Glucans from various sources has a wide range of potential applications in food production such as thickening agent, fat substitute, and emulsifier [4]. Particularly, β-glucan draws attention as an immunostimulant for human use [1, 3, 9]. Yeast cell wall is one of the most common sources for β-glucan production. Some studies showed that β-glucan from brewer’s yeast exhibits good ability to improve immune system [11]. β-Glucans isolated from baker’s or brewer’s yeast can be used in the production of salad toppings (dressings), frozen deserts, sauces, yogurts and other milk products, softdoughs and paning doughs, conditories and mixture for cake filling (Seeley, 1977; Read and Nagodawithana, 1991). The ability of β-glucan to retain water can be also used in the production of sausages and other meat products (Th ammakiti et al., 2004). Its gelling, water-holding and oil-binding characteristics make it suitable for many food products (Reed and Nagodawithana, 1991; Lazzari, 2000; Wylie-Rosett, 2002; Th ammakiti et al., 2004), such as the production of mayonnaise and sausages [13]. The possible use of yeast β-glucans in the different food products is illustrated in table 1.



Table 1. The application of β-glucan in food products [13]

Spent brewer’s yeast is produced in huge amounts as a secondary product in breweries all around the world. Most of it is usually sold after heat inactivation as a cheap feed supplement. The rest of it ends in waste water disposal and pollutes the natural water sources with organic material (Thammakiti et al., 2004; Seeley, 1977; Suphantharika et al., 2003; Thammakiti et al., 2004; Liu et al., 2008) [15].

β-Glucan preparations extracted from spent brewer’s yeast is the way to obtain high valuable product from a cheap raw-material (Worrasinchai et al., 2005), that showed high apparent viscosity, water holding, oil binding, and emulsion stabilizing capacities (Thammakiti et al., 2004) and could be used in food products as a thickener and fat replacer. Few authors such as Worrasinchai et al. (2006), Santipanichwong and Suphantharika (2007) and Satrapai and Suphantharika (2007) performed their research using β-glucan isolated by Thammakiti et al. (2004) and applied it later in different food systems (for example mayonnaises with reduced fat amounts) [12].

There are some researches mentioned to extraction of beta-Glucan from Brewer’s Yeast [14]. Vesna Zechner-Krpan et al. (2009) conducted two different procedures to isolate water-insolubleb-glucans from brewer’s yeast: alkaline-acidic isolation (AA) and alkaline-acidic isolation with mannoprotein removal (AAM). The obtained beta-glucans were then dried by air-drying, lyophilization and combination of sonication and spray-drying. b-Glucan preparations obtained by AA and AAM isolations had similar values of dry mass, total polysaccharides, proteins and organic elemental microanalysis. The mass fractions ofb-glucan in total polysaccharides were significantly affected by different isolation procedures. Fourier transform infrared (FTIR) spectra of all preparations had the appearance typical for (1→3)-β-D-glucan. Lyophilization and especially air-drying caused a higher degree of agglomeration and changes in β-glucan microstructure. Sonication followed by spray-drying resulted in minimal structural changes and negligible formation of agglomerates [13]. Vesna Zechner-



Krpan et al. (2010) investigated three different drying methods were used: air-drying, lyophilization and spray-drying. Air-drying and lyophilization caused β-glucan particles agglomeration and changes of their microstructure. Sonication combined with spray-drying resulted in minimal β-glucan structural changes and negligible formation of agglomerates. Reaggregation of spray-dried β-glucan particles was minimal even after resuspending in water [15].

The purpose of the present study was to find out the optimum conditions for β-glucan extraction from S. cerevisiae cell wall by autolysis, enzyme, ultrasound and combined enzyme-ultrasound methods.

2. EXPERIMENTAL

2.1. Materials

Spent brewer’s yeast slurry (a strain of S. cerevisiae), a by-product from brewery, was provided by Saigon Beer Alcohol Beverage Corporation (SABECO), Ho Chi Minh City, Vietnam. Alcalase® 2.4 LFG was obtained from Novozymes Co. All other chemicals were of analytical grade.

2.2. Procedures for preparing β-glucan from spent yeast

Spent brewer’s yeast was washed in distilled water using a 1:3 of the yeast slurry: water (g/g) ratio. Natural sedimentation was performed for 1 hour and followed by decantation step. The sediment was centrifuged at 3000 rpm for 15 minutes for wet yeast biomass recovery. Wet biomass was kept in cool condition (4oC) for 2-3 days. Biomass was adjusted to 15% (w/w) suspension in phosphate-citrate buffer (pH 7.0) and subjected to the cell wall disruption step assisted by enzyme and ultrasound. The acquired mixtures was heated to 121oC by autoclave for 4 hours and centrifuged at 3000 rpm for 15 minutes to separate the liquid phase. The insoluble residues were washed twice with distilled water. The obtained cell walls were suspended in organic solvent with solvent: solid ratio of 1:4 (w/w). The wet β-glucan residues were centrifuged and dried at an air temperature of 40oC for 1 hour.

2.3. Optimization of cell wall disruption

Table 2. Core value and disruption steep in autolysis cell wall disruption

Coefficents Time of autolysis X1 (h) pH X2

Core value 30 5

Disruption step 5 0.5



To investigate the optimum conditions of β-glucan extraction assisted by enzyme and ultrasound, the rotatable central composite design with a quadratic model was selected (Table ). The model is expressed as Eq. (1):

Y = b0 + b1X1+ b2X2 + b12X12 + b11X12 + b22X2

2 (1)

where Y = response, X1 and X2 = coded variables, b = estimated coefficients in the response surface model.

Table 3. Independent variables and their coded and actual values used in response surface design of enzyme and ultrasound-assisted condition

Coded levels Independent variable Symbol

–1 0 +1

Enzyme concentration (% w/w)

Treatment time (hour)

Sonication power (W/g)

Treatment time (minute)

X1

X2

X3

X4

0.5

2

6.5

8

0.75

4

7.5

10

1

6

8.5

12

2.4. Analytical methods

The β-glucan content in the preparations was determined enzymatically by the commercial assay ‘‘YEAST BETA GLUCAN ASSAY KIT’’ (Megazyme Int., Bray, Ireland). Crude protein content of the cell wall fractions was determined by a Kjeldahl method. Soluble protein content was determined according to the procedure described by Lowry (1951) [6].

2.5. Calculation of cell wall disruption yield

The cell disruption yield was defined as the ratio of soluble protein content in the extract to total protein content in the yeast slurry. β-glucan = Total glucan − α-glucan − oligomer[7] Cell wall discruption yield:

100

BT

BD

PP

PP=H [8]

Where: PD: soluble protein after cell wall disruption. PB: soluble protein before cell wall disruption. PT: total protein.



3. RESULTS AND DISCUSSION

3.1 Autolysis cell wall extraction

Table 4. The regression equation of autolysis cell wall extraction

Test run no X0 X1 X2 X1X2 Y

1 +1 -1 -1 +1 43.97

2 +1 +1 -1 -1 45.20

3 +1 -1 +1 -1 42.74

Test run at core: 2k

4 +1 +1 +1 +1 38.88

5 +1 -√2 0 0 45.33

6 +1 +√2 0 0 41.82

7 +1 0 -√2 0 44.33

Test run at cross points with two axis: 2k

8 +1 0 +√2 0 40.66

9 +1 0 0 0 49.13

10 +1 0 0 0 52.51

Test run at core

11 +1 0 0 0 51.79

Table 5. the regression coefficients of autolysis cell wall extraction

Coefficient Value Standard deviation

P P < 0.05

b0 51.1434 0.727583 1.10364E-008 Accept

b1 -0.949285 0.445586 0.086358 Not accept

b2 -1.59266 0.445586 0.0159705 Accept

b11 -3.86913 0.530421 0.000757944 Accept

b22 -4.4093 0.530421 0.000411602 Accept

b12 -1.2725 0.630105 0.0994332 Not accept

R2 = 0.959, Q2 = 0.867. The regression equation: Y= 51.14-1.59X2-3.87X1

2-4.41X22

On 3-dimension scale, Y = 51.32%, X1 = 29.53 (h) and X2 = 4.92.

3.2 Optimization of enzyme and ultrasound-assisted extraction

Table 6 shows the matrix of central composite design (22) with the level of variation and the dependent variable Y (experimental response) expressed as % cell wall disruption for enzyme and ultrasound-assisted extraction.



Table 6. Experimental design matrix used in response surface design of enzyme and ultrasound-assisted extraction

Coded level of variable Response 2c Test run a no.

X1 X2

Response 1b

1

2

3

4

5

6

7

8

9

10

11

–1

+1

–1

+1

–1.14

+1.14

0

0

0

0

0

–1

–1

+1

+1

0

0

–1.14

+1.14

0

0

0

25.37

36.75

43.73

47.77

38.25

42.63

20.23

45.57

45.06

45.20

43.36

23.81

29.06

32.04

41.58

29.93

40.42

28.33

39.85

39.52

39.11

39.71 a Test runs were performed in random order. b Response 1 was expressed as disruption yield obtained in enzyme-assisted extraction. c Response 2 was expressed as disruption yield obtained in ultrasound-assisted extraction.

The results of multiple regression analysis of two independent variables on response along with the results of analysis of variance (ANOVA) were summarized in Table . The regression analysis showed a significant probability of P-value (p< 0.05) in estimating cell wall disruption yield, which means that the two independent variables had significant effects on response.

Table 7. Statistical significance obtained for the regression coefficients of the enzyme and ultrasound-assisted extraction

Coefficient Regression coefficient P value

b0

b1

b2

b11

b22

b12

Regression

R square

Q square

44.54 (39.45)

2.70 (3.71)

8.15 (4.63)

–1,62 (–2.89)

–5.39 (–3.43)

–1.84 (1.07)

0.970 (0.947)

0.797 (0.628)

<0.01a (<0.01a)

0.02 (<0.01a)

<0.01a (<0.01a)

0.14 (0.02)

<0.01a (0.01)

0.15 (0.34)

a Significant at 1% level (p< 0.01).

Value in parentheses is of the ultrasound-assisted extraction.

The correlation coefficient, R2, which is a measure of how well the model can be made to fit the raw data, was more than 0.90, indicating an adequate model fit.



Figure 3. Response surface for the effects of (a) enzyme and (b) ultrasound-assisted extraction conditions on the cell wall disruption yield.

The model for enzyme-assisted extraction could be expressed as:

Y = 44.54 + 2.70X1 + 8.15X2 – 5.39X22 (2)

The set of optimum conditions was obtained by the use of RSM optimization and was as follows: enzyme concentration of 0.86% (w/w) and treatment time of 5.34 hours. Under the conditions, the predicted and practical response value were 47.92% and 46.79%, respectively.

The model for ultrasound-assisted extraction could be expressed as:

Y = 39.45 + 3.70X1 + 4.63X2 – 2.89X12 – 3.43X2

2 (3)

The optimum conditions were as follows: ultrasound power of 8.29 W/g and extraction time of 11.60 minutes. The predicted cell wall disruption yield under the optimum condition was 42.76%. The experimental value of 42.55%, obtained from practical experiments, demonstrated the validation of the RSM model, indicating that the model was adequate for the extraction process.

3.3 The combined method for cell wall disruption

To improve the yeast disruption, we performed tests using combination of enzyme and ultrasound treatment. The cell wall disruption yield had maximum value (58.56%) in case of enzyme hydrolysis followed by sonication. When this step was carried out in the reverse order, the lower value was observed (51.95%). 8 summarized the composition of β-glucan preparations in various methods.



Table 8. Composition of the β-glucan preparation in various methods

Sample Water (%) Protein (%) Lipid (%) Ash (%) β-glucan (%)

Control 78.23ab±1.52 52.72a±2.08 3.53d±0.37 6.50d±0.65 26.92a±1.96

Ultrasound 79.64bc±0.61 26.53b±0.92 2.50c±0.28 5.22b±0.62 56.50b±1.28

Enzyme 77.38a±1.16 23.33c±1.18 1.33a±0.13 3.62a±0.21 60.32c±1.01

Ultrasound-enzyme

80.63c±0.71 20.20d±1.12 1.25a±0.09 3.43a±0.18 65.43d±1.93

Enzyme-ultrasound

79.48bc±1.20 15.38e±1.34 1.10a±0.07 3.48a±0.31 72.06e±1.23

Components based on dry basis.

Values which have the same superscript symbol in the same column have no significant difference (p= 0.05).

Due to yeast cell wall’s rigidity, ultrasonic treatment is not capable of breaking down it [7]. However, after hydrolyzed by enzyme, yeast cell became easy to be disrupted. Therefore, the highest yield in samples subjected to enzymatic hydrolysis followed by sonication can be inferred from that. Prior enzymatic hydrolysis decomposing mannoprotein component and reducing the regidity of cell wall could support ultrasound in further disruption. The higher extraction yield, the higher β-glucan and lower other components concentration.

4. CONCLUSION

A considerable history of safe use of beta-glucans also exists through the consumption of yeast, cereals and mushrooms among other foods, as well as food supplements. As a result of extensive research, one could expect increasing application of β-glucan in food production in the near future. β-Glucan obtained from spent brewer’s yeast possesses properties that are beneficial for food production. The use of spent brewer’s yeast for isolation of β-glucan intended for food industry would represent a payable technological and economical choice for breweries. With the aim of preserving the β-glucan structure and bioactivity, enzyme and ultrasound-assisted extraction are among the potential methods. Moreover, the cell wall disruption yield can be improved in case of combination of these methods in appropriate order. The yeast suspensions which are subjected to enzyme treatment followed by ultrasonic shows good result with cell wall disruption yield of 58.56% and β-glucan content of 72.06% on the dry basis.




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ΒETA-GLUCAN EXTRACTION FROM WASTE … SACCHAROMYCES CEREVISIAE USING AUTOLYSIS, ENZYME, ULTRASONIC...

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